Chemokines play a critical role in the trafficking of monocytes and macrophages in vascular disease, but the molecular mechanisms of their actions are poorly understood. Fractalkine is a novel chemokine with an unusual architecture. Unlike soluble chemokines, fractalkine consists of a chemokine-like domain fused to a membrane-bound mucin stalk. A soluble form of fractalkine is created by the action of TACE, a metalloprotease that cleaves fractalkine just above the transmembrane domain. In its soluble form fractalkine is a potent chemoattractant, but in its membrane-bound form it captures cells bearing its cognate receptor, CX3CR1. We have shown that mice in which CX3CR1 is deleted by gene targeting are protected against rejection of heterotopic cardiac transplants, and diet-induced atherosclerosis. However, the relative contributions of the membrane-bound and soluble forms of fractalkine to these vascular disease are unknown. To investigate the role of membrane-bound FK we will create knock-in mice expressing a noncleavable form of fractalkine. To investigate the role of soluble fractalkine, we will create mice that express soluble, but not membrane-bound FK. These studies will test the hypothesis that the cleavage of FK contributes to atherogenesis. Recent genetic studies in patients have revealed that a polymorphism in CX3CR1 correlates with protection from coronary artery disease. We will introduce this V259I/T280M mutation into murine CX3CR1, and test the hypothesis that the polymorphism affords protection from atherosclerosis by preventing the capture of monocyte/macrophages. Fractalkine is not the only chemokine that has been found to play a role in atherosclerosis. We have previously shown that MCP-1, and its receptor CCR2, contribute to macrophage recruitment and lesion formation. In the final portion of this proposal, we will create double knockout mice lacking both fractalkine and CCR2 to determine if these chemokines act independently or in concert to promote atherogenesis. Completion of these aims will significantly advance our understanding of the mechanisms by which fractalkine contributes to lesion formation in atherosclerosis.